Fig 2: AMPK is required for Beclin1 phosphorylation in phosphorus-induced lipophagy in tilapia hepatocytes. Tilapia hepatocytes were incubated with Pi and AMPKa1-knockdown experiment for 48 h in L-15 medium. (A) The presence of Lyso-Tracker Red-stained autolysosomes was demonstrated by the flow cytometry, showing the mean fluorescence intensity. (B) The Lyso-Tracker Red (50 nM)-stained autolysosome was calculated by the flow cytometric analysis of the mean fluorescence intensity. (C) The presence of AO-stained autophagic vacuole was demonstrated by the flow cytometry analysis of the mean fluorescence ratio. (D) The AO (1 µM)-stained autophagic vacuole was calculated by the flow cytometric analysis of the mean fluorescence ratio. (E) Representative confocal microscopy image of hepatocytes stained with acridine orange. (F, G) Western blot analysis and quantification analysis of the Beclin1, p-Beclin1, p62 and LC3B protein levels. (H) TG content. (I) The presence of LDs with Bodipy 493/503 staining was demonstrated by the flow cytometry of tilapia hepatocytes. (J) Bodipy 493/503-stained LDs was quantified by the flow cytometric analysis of FL1 (green) mean fluorescence intensity of tilapia hepatocytes. (K) Representative confocal microscopy image of tilapia hepatocytes with Bodipy 493/503 staining. All data are mean ± SEM (n = 3), *P = 0.05, **P = 0.01, ***P = 0.001, the P Value was calculated by the one-way ANOVA with the Bonferroni post-hoc test.

Fig 3: Phosphorus incubation activated AMPK pathway in tilapia hepatocytes. (A) Relative mRNA abundances of genes related to AMPK pathway. (B,C) Western blot analysis and quantification analysis of p-AMPKa1 protein levels. All data are expressed as mean ± S.E.M (n = 3). P value was calculated by the Student's t tests. *P = 0.05, **P = 0.01, compared with the control.

Fig 4: Phosphorylation of Beclin1 by AMPK is necessary for Pi-induced autophagy to degrade LD. (A) Alignment of the partial Beclin1 protein sequences among several species. Red S indicates putative serine sites where Beclin1 could be phosphorylated. (B) Alignment of partial AMPKa1 protein sequences among several species. Red S indicates putative serine sites where AMPKa1 could be phosphorylated. (C) The interaction between AMPKa1 and Beclin1. His-tag AMPKa1 and GFP-tag Beclin1 were transfected into the HEK293T cells. (D) Mutations of S496A reduced the Beclin1 phosphorylation level. Phosphorylation of the ectopically expressed WT and S496A was analyzed. (E) Mutations of S90A decreased the AMPKa1 phosphorylation level. Phosphorylation of the ectopically expressed WT and S90A was analyzed. (F) The interaction between AMPKa1 and Beclin1 after Beclin1 and AMPKa1-overexpression and Pi treatment.

Fig 5: Dietary phosphorus supplementation activated the AMPK pathway and increased AMPKa1 phosphorylation level in tilapia liver. (A) Relative mRNA levels of genes related to AMPK pathway. (B,C) Western blot analysis of p-AMPKa1 protein levels. Data are mean ± SEM (n = 3). “a–c” denote significance at P = 0.05. The P value was calculated by the one-way ANOVA and further the post-hoc Duncan's multiple range testing.